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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Cancer Res. 2011 Mar 9;71(9):3400–3409. doi: 10.1158/0008-5472.CAN-10-0965

Phosphoglucose Isomerase/Autocrine Motility Factor mediates epithelial-mesenchymal transition regulated by miR-200 in breast cancer cells

Aamir Ahmad 1, Amro Aboukameel 2, Dejuan Kong 1, Zhiwei Wang 1, Seema Sethi 1, Wei Chen 2, Fazlul H Sarkar 1,*,#, Avraham Raz 1,2,#
PMCID: PMC3085607  NIHMSID: NIHMS279326  PMID: 21389093

Abstract

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) plays important role in glycolysis and gluconeogenesis, and is associated with invasion and metastasis of cancer cells. We have previously shown its role in the induction of Epithelial-to-Mesenchymal transition (EMT) in breast cancer cells, which led to increased aggressiveness; however, the molecular mechanism by which PGI/AMF regulates EMT is not known. Here we show, for the first time, that PGI/AMF over-expression led to an increase in the DNA-binding activity of NF-κB, which, in turn, led to increased expression of ZEB1/ZEB2. The microRNA-200s (miR-200s; miR-200a, miR-200b and miR-200c) are known to negatively regulate the expression of ZEB1/ZEB2, and we found that the expression of miR-200s was lost in PGI/AMF over-expressing MCF-10A cells as well as in highly invasive MDA-MB-231 cells, which was consistent with increased expression of ZEB1/ZEB2. Moreover, silencing of PGI/AMF expression in MDA-MB-231 cells led to over-expression of miR-200s, which was associated with reversal of EMT phenotype i.e. Mesenchymal-to-Epithelial Transition (MET), and these findings were consistent with alterations in the relative expression of epithelial (E-cadherin) and mesenchymal (vimentin, ZEB1, ZEB2) markers, and decreased aggressiveness as judged by clonogenic, motility and invasion assays. Moreover, either re-expression of miR-200 or silencing of PGI/AMF suppressed pulmonary metastases of MDA-MB-231 cells in vivo, and anti-miR-200 treatment in vivo resulted in increased metastases. Collectively, these results suggest a role of miR-200s in PGI/AMF induced EMT, and thus approaches for up-regulation of miR-200s could be a novel therapeutic strategy for the treatment of highly invasive breast cancer.

Keywords: Phosphoglucose Isomerase, Autocrine Motility Factor, microRNA-200s, Epithelial-Mesenchymal Transition (EMT), Breast Cancer, Invasion

Introduction

Phosphoglucose Isomerase (PGI) is a housekeeping cytosolic enzyme that brings the inter-conversion of glucose-6-phosphate and fructose-6-phosphate (1) playing key role during glycolysis and glucogenesis. Additionally, PGI serves several other functions – as tumor-secreted cytokine, autocrine motility factor (AMF), it stimulates cell motility, migration, invasion and metastasis (2, 3); as neurotrophic factor, neuroleukin, it supports the survival of embryonic spinal neurons, skeletal motor neurons and sensory neurons (4); and, as maturation factor, it mediates differentiation of human myeloid leukemic cells (5). Other functions of PGI include its role as sperm antigen-36 (6) and a serine proteinase inhibitor (7). PGI/AMF and its receptor are also associated with cancer progression and poor prognosis (8-10).

Recent evidences have suggested that PGI/AMF plays an important role during the intra-conversion between EMT and MET (11-13). The phenomenon of EMT is associated with acquisition of invasive phenotype by cancer cells (14, 15), and, in particular, an aggressive behavior of breast cancer cells in vitro (16) and in vivo (17). The role of PGI/AMF in the progression of breast cancer is well established (10, 18, 19), which is consistent with our recent observation (12) showing induction of EMT by PGI/AMF in MCF-10A non-tumorigenic human breast epithelial cells. Conversely, we found reversal of EMT leading to mesenchymal-to-epithelial transition (MET) upon silencing of PGI/AMF in aggressive MDA-MB-231 cells. These findings suggested an importance of PGI/AMF in the regulation of EMT; however, the molecular mechanism is yet to be established.

Loss and gain of specific microRNAs (miRNAs) is associated with invasion and metastasis, and miRNAs are known to regulate the acquisition of cells' mesenchymal phenotype (20-22). However, nothing is known as to the interrelationships between PGI/AMF, miRNAs and EMT-MET, which prompted the current investigation. Here we report for the first time that PGI/AMF over-expression could lead to increased DNA binding activity of NF-κB, which transcriptionally up-regulates the expression of ZEB1 and ZEB2, resulting in the induction of EMT, associated with the loss of miR-200s expression.

Materials and Methods

Cell lines and Reagents

MCF-10A human breast epithelial cells were maintained in DMEM-F12 medium supplemented with 0.1 μg/mL cholera toxin, 0.02 μg/mL epidermal growth factor, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5% horse serum (12). Human breast cancer cell lines MDA-MB-231 and BT-549 were cultured in DMEM and RPMI media, respectively, with 10% fetal bovine serum and penicillin/streptomycin. All cells were cultured in 5% CO2–humidified atmosphere at 37°C. The cell lines have been tested and authenticated in core facility (Applied Genomics Technology Center at Wayne State University) by short tandem repeat profiling using the PowerPlex 16 System from Promega. Antibodies were purchased from following sources - vimentin (Abcam, Cambridge, MA), MMP-9 (R&D Systems, Minneapolis, MN), E-cadherin, ZEB1 and uPAR (Santa Cruz Biotechnology Inc., Santa Cruz, CA), ZEB2 and β-actin (Sigma-Aldrich, St Louis, MO).

Real-Time RT-PCR

Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Real-time PCR was used to quantify mRNA expression. Sequences of primers for E-cadherin, vimentin, ZEB1, ZEB2 and GAPDH were same as reported earlier (23) and the amount of RNA was normalized to GAPDH expression. For miRNA analysis, total RNA was isolated using the mirVana miRNA isolation kit (Ambion). The levels of miRNAs were determined using miRNA-specific Taqman MGB probes from the Taqman MicroRNA Assay (Applied Biosystems). The relative amounts of miRNA were normalized to RNU6B.

Transfection Experiments

Detailed methodology for the generation of stably transfected MCF-10A cells with full-length PGI/AMF cDNA and MDA-MB-231 cells with specific small interfering RNA (siRNA) targeting PGI/AMF has been described (12). Cell clones were maintained by adding 300μg/mL zeocin (Invitrogen) to the culture medium.

Preparation of nuclear lysates and Electrophoretic Mobility Shift Assay (EMSA)

Nuclear protein extract was prepared and subjected to EMSA for assessing the DNA binding activity of NF-κB (24). EMSA was performed by incubating 4μg of nuclear protein extract with IRDye-700–labeled nuclear factor-κB (NF-κB) oligonucleotides (LI-COR, Lincoln, NE). Incubation mixture included 2μg of poly dI-dC (poly deoxyinosinic-deoxycytidylic acid) in the binding buffer. DNA-protein complex formed was separated from free oligonucleotide on 8% native polyacrylamide gel and then visualized by Odyssey Infrared Imaging System using Odyssey Software Release 1.1 (Li-COR, Inc., Lincoln, NE).

Wound healing assay

Monolayer cultures of MCF-10A and MDA-MB-231 (control and PGI/AMF-transfected/silenced), seeded in 6-well plates, were wounded with 200 μl pipette tips once they were 80% confluent. Pictures were taken at 0 and 24 h time points under phase contrast microscope to monitor the migration of cells into the open space (wound). Quantification of wound healing was done using NIH Image-J software. The outlines of wound were marked by two diagonal parallel lines in each image. Lines within the wound mark the progress of cells that migrated into the wound. The migratory distances (in microns) of all cells that migrated into the wound were totaled and are represented as bar graphs.

Clonogenic Assay

To test the survival of breast cells, clonogenic assay was performed (25). Briefly, cells were plated in six-well plates, incubated overnight, appropriately transfected, grown for 48 hours, trypsinized, and the viable cells counted (trypan blue exclusion) and plated in 100 mm petri dishes in a range of 100 to 1000 cells per plate. The cells were then incubated for 14 to 21 days at 37°C in a 5% CO2/5% O2/90% N2 incubator and colonies were stained with 2% crystal violet, counted and photographed.

Western blot analysis

For Western blot analysis, cells were lysed in RIPA buffer containing complete mini EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich, St. Louis, MO) (24). After resolution on 12% polyacrylamide gels under denaturing conditions, proteins were transferred to nitrocellulose membranes, incubated with appropriate primary / horseradish peroxidase-conjugated secondary antibodies and visualized using chemiluminescence detection system (Pierce, Rockford, IL).

Cell invasion and migration assay

Cell invasion assay was performed using 24 well Transwell Permeable Supports with 8 μM pores (Corning, Lowell, MA) (25). Cells were suspended in serum free medium and seeded into the Transwell inserts coated with growth factor reduced Matrigel (BD Biosciences, Bedford, MA). Bottom wells were filled with media containing complete media. After 24 h, cells were stained with 4 μg/ml calcein AM (Invitrogen) in PBS at 37°C for 1 h and photographed under a fluorescent microscope. The cells were detached from inserts by trypsinization and fluorescence of the invaded cells was read in ULTRA Multifunctional Microplate Reader (TECAN, San Jose, CA).

Experimental pulmonary metastasis assay

The animal experimental protocol was approved by the Committee on the Ethics of Animal Experiments of Wayne State University Institutional Users of Animal Care Committee. Two million MDA-MB-231 cells transfected with either empty-vector or PGI/AMF-specific siRNA (PGI/AMF-silenced cells), were injected through tail vein of female ICR-SCID (5-6 weeks old) mice obtained from Taconic Farms. Effect of pre-miR-200s was studied by transfections of expression plasmids with pre-miR-200s (Origene Technologies, Rockville, MD) in MDA-MB-231 vector control cells prior to injection in mice. To study the effect of anti-miR-200s in vivo, PGI/AMF-silenced MDA-MB-231 cells were injected in mice via tail vein, and starting with the next day, animals were treated with PBS-formulated locked-nucleic acid (LNA)-modified oligonucleotide (LNA-anti-miR200b) (obtained from Exiqon, Woburn, MA) (26) administered via intravenous injections (25 mg/kg) three times a week, for a total of 5 weeks. All mice were euthanized at the end of week 5. Lungs were removed, formalin fixed and stained with hematoxylin and eosin (H & E). Lung metastatic nodules were counted microscopically. Histological and microscopic analysis was carried out by a pathologist and the data was analyzed by a statistician.

Data analysis

The experimental results presented in the figures are representative of three or more independent observations. The data are presented as the mean values ± SE. For in vivo group comparison tests, we used log-transformed data with continuity correction. The normality assumption in each group was checked using Kolmogorov-Smirnov test. The pair-wise two-sample t test is used if normality assumption holds. The pair-wise Wilcoxon test is used otherwise if normality assumption fails. Values of p < 0.05 were considered to be statistically significant.

Results

Expression of PGI/AMF correlates with ZEB1/ZEB2 and NF-κB activity

It has been reported that ectopic expression of PGI/AMF in non-tumorigenic cell line MCF-10A results in an increase in the expression of mesenchymal marker vimentin, accompanied by a decrease in epithelial marker E-cadherin (12), suggesting the acquisition of EMT phenotype. Here we performed RT-PCR assessing the mRNA transcripts of these markers as well as other mesenchymal markers, ZEB1 and ZEB2. In addition to the verification of earlier results with E-cadherin and vimentin (Fig. 1A), we found that PGI/AMF transfection resulted in a significant increase in the expression of ZEB1 (4-fold increase) as well as ZEB2 (60% increase) (Fig. 1A; p<0.01). Down regulation of PGI/AMF expression in MDA-MB-231 resulted in an increase in E-cadherin mRNA expression concomitant with a decreased vimentin expression (Fig. 1A), A significant decrease in the expression of ZEB1 (almost 100% inhibition) and ZEB2 (62% increase) (Fig. 1A) in these cells (p<0.01) was also noted. To rule out any cell line-specific effects, we carried out similar studies using aggressive BT-549 breast cancer cells and found that the silencing of PGI/AMF resulted in a significant increase in E-cadherin and decrease in vimentin/ZEB1/ZEB2 expression (Fig.1B), which was consistent with the data obtained from MDA-MB-231 cells.

Figure 1.

Figure 1

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Effect of PGI/AMF expression on markers of EMT. Expression of E-cadherin, vimentin, ZEB1, ZEB2 was evaluated by Real-Time RT-PCR in (A) MCF-10A and MDA-MB-231 cells, and (B) BT-549 cells. (C) Effect of PGI/AMF expression on DNA-binding activity of NF-κB. Nuclear proteins were subjected to gel shift assay and densitometric analysis of principal NF-κB bands (marked by arrows) is represented in lower panel. Intensity of NF-κB band in the control cells was taken as 1 and the relative intensities of PGI/AMF-transfected/silenced cells are plotted. (D) Effect of PGI/AMF expression on NF-κB target genes, uPAR and MMP-9 was evaluated by western blot analysis. β-actin protein was used as protein loading control. V, vector control; P/A, PGI/AMF-transfected cells; sP/A, PGI/AMF-silenced cells. **p<0.01 vs. vector control.

It is known that ZEB1/ZEB2 can be regulated by NF-κB (27) and that cells undergoing EMT have elevated NF-κB activity (28, 29). Therefore, we evaluated DNA-binding activity of NF-κB in our models (Fig 1C). The characteristic p65 NF-κB bands were quantitated (identified by the arrows in the figure) and we found increased NF-κB activity (more than 4-folds) in MCF-10A cells transfected with PGI/AMF and also in control MDA-MB-231 and BT-549 cells that have natural mesenchymal phenotype. We also observed a significant decrease in the DNA binding activity of NF-κB in MDA-MB-231 and BT-549 cells (47% and 50% respectively) silenced for PGI/AMF expression (Fig. 1C). These observations suggested a plausible involvement of NF-κB signaling in EMT-induction by PGI/AMF. To confirm this, we evaluated the expression of uPAR and MMP-9, two NF-κB downstream target genes, and found that ectopic expression of PGI/AMF in MCF-10A cells led to increased expression of uPAR and MMP-9, whereas silencing of PGI/AMF in MDA-MB-231 and BT-549 cells led to a significant down-regulation of their expression (Fig. 1D). uPAR (30) and MMP-9 (31) have been linked with the induction of EMT in breast cancer cells which further implicates the role of NF-κB signaling in PGI/AMF-induced EMT. Silencing of PGI/AMF in MDA-MB-231 and BT-549 cells showed similar effects on markers of EMT and NF-κB signaling. Therefore, we chose MDA-MB-231 cells for further detailed mechanistic studies.

Increased PGI/AMF expression reduced miR-200s, and re-expression of miR-200s restored epithelial phenotype in MCF-10A cells

In addition to the activation by NF-κB, ZEB1/ZEB2 are also regulated by miR-200 family (22, 32, 33). Therefore, we questioned whether the levels of miR-200 family could be affected in PGI/AMF-expressing MCF-10A cells. Analysis of basal levels of miR-200 family in the paired cell lines revealed that the over-expression of PGI/AMF caused a significant decrease (miR-200a - 60%; miR-200b − 38% and miR-200c − 20%) in the levels of all three miR200s (Fig. 2A). To assess functional implications of these findings, we transfected PGI/AMF over-expressing MCF-10A cells with a cocktail of pre-miRNAs (pre-miR-200a + pre-miR-200b + pre-miR-200c). Transfection of pre-miRNAs is a standard technique to induce the expression of target miRNAs (23). Re-expression of miR-200s resulted in increased expression of E-cadherin (2.5-folds increase) (Fig. 2B; p<0.01) and decreased expression of vimentin (56% decrease) (Fig. 2C; p<0.01), ZEB1 (79% decrease) (Fig. 2D; p<0.01) as well as ZEB2 (25% decrease) (Fig. 2E; p<0.05). The levels of miRNAs in pre-miRNA-transfected cells approached those in native cells thus verifying the efficiency of re-expression of miRNAs (Fig. 2A).

Figure 2.

Figure 2

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Re-expression of miR200s reverses EMT induction by PGI/AMF in MCF-10A cells. (A) Basal expression of miR-200a, miR-200b and miR-200c was evaluated by Real-Time RT-PCR in MCF-10A (vector control vs. PGI/AMF expressing cells ± pre-miRNAs). (B-E) Effect of re-expression of miR200s (miR-200a+miR-200b+miR200c) in PGI/AMF expressing MCF-10A cells on the expression of indicated EMT markers was also evaluated by Real-Time RT-PCR. N, non-specific pre-miRNA; PM or M, specific pre-miRNAs (pre-miR200a + pre-miR200b + pre-miR200c) *p<0.05 and **p<0.01 vs. vector / non-specific control.

PGI/AMF silencing-induced MET could be reversed by suppressing the expression of miR-200s

Induction of MET by silencing of PGI/AMF in aggressive MDA-MB-231 cells has been previously demonstrated (12). Here we found that such silencing results in an increased expression of all miR-200s (miR200a − 3.5-folds; miR200b − 5.8-folds and miR200c − 2.7-folds) (Fig. 3A; p<0.01). Among the three miR-200s, miR-200b levels were found to be particularly elevated. Suppression of these up-regulated miR-200s, using a cocktail of specific anti-miR-200s (anti-miR-200a + anti-miR-200b + anti-miR-200c) resulted in de-repression of vimentin mRNA transcripts (Fig. 3B; p<0.01). Levels of miRNAs in anti-miRNA-transfected cells approached those in native cells, thus verifying the efficiency of down-regulation of miRNAs (Fig. 3A). Expression of E-cadherin, which was elevated in PGI/AMF-silenced cells, was found to be inhibited by anti-miR-200s (Fig. 3C; p<0.01) while the expression of ZEB1 and ZEB2 was found to be concomitantly de-repressed by anti-miR-200s (Fig. 3D and E; p<0.01).

Figure 3.

Figure 3

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Suppression of miR200s negates PGI/AMF-silencing-induced effects on EMT in MDA-MB-231 cells. (A) Basal expression of miR-200a, miR-200b and miR-200c was evaluated by Real-Time RT-PCR in MDA-MB-231 (vector control vs. PGI/AMF-silenced cells ± anti-miRNAs). (B-E) Effect of suppression of miR200s (miR-200a+miR-200b+miR200c) in PGI/AMF-silenced MDA-MB-231 cells on the expression of EMT markers was also evaluated by Real-Time RT-PCR. N, non-specific anti-miRNA; AM or M, specific anti-miRNAs (anti-miR200a + anti-miR200b + anti-miR200c) **p<0.01 vs. vector / non-specific control.

Role of miR-200s in cell motility and clonogenicity of breast cells

In MCF-10A cells transfected with PGI/AMF, significant migration of cells into open space (wound) was observed after 24 h, which was found to be inhibited by re-expression of miR-200s (Fig. 4A). In MDA-MB-231 cells, wound closure was observed in the control cell cultures after 24 h but not in PGI/AMF-silenced cells (Fig. 4B). Expression of miR-200s in control cells inhibited wound healing whereas suppression of miR-200s, by anti-miR200s, in PGI/AMF-silenced cells stimulated cell motility (Fig. 4B).

Figure 4.

Figure 4

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miR-200s influence motility and clonogenicity of breast cells. (A) MCF-10A cells and (B) MDA-MB-231 cells were wounded (time 0h) and maintained for 24h in normal medium. (C) Anchorage-dependent assays for clonogenicity were performed as described under “Methods” section. Bar graphs on the right represent quantification of results presented on the left, in each case. V, vector control; P/A, PGI/AMF-transfected cells; sP/A, PGI/AMF-silenced cells, NP, non-specific pre-miRNA; PM, specific pre-miRNAs (pre-miR200a + pre-miR200b + pre-miR200c); NA, non-specific anti-miRNA; AM, specific anti-miRNAs (anti-miR200a + anti-miR200b + anti-miR200c). *p<0.05 and **p<0.01 vs. non-specific control, NS, non-significant.

Next, we tested the effect of PGI/AMF and miRNA-200s on the clonogenic potential of MCF-10A and MDA-MB-231 cells. PGI/AMF-transfected MCF-10A cells formed more colonies as revealed by crystal violet staining, and re-expression of miR-200s suppressed this effect (82% decrease) (Fig. 4C). Silencing of PGI/AMF in MDA-MB-231 cells, on the other hand, significantly decreased the number of colonies (57% decrease) and this inhibition was reversed by the suppression of miR-200s (∼3-folds increase in number of colonies) (Fig. 4C).

miR-200s reverse the effects of PGI/AMF on markers of EMT and invasion of breast cells

Re-expression of miR-200s in PGI/AMF-transfected MCF-10A cells resulted in re-expression of E-cadherin and suppression of vimentin/ZEB1/ZEB2 while suppression of miR-200s caused a down-regulation of E-cadherin and up-regulation of vimentin/ZEB1/ZEB2 in PGI/AMF-silenced MDA-MB-231 cells (Fig. 5A). Since PGI/AMF is involved in the invasion of breast cancer cells, we performed invasion assays using matrigel-coated transwell inserts (Fig. 5B). In MCF-10A cells, PGI/AMF-induced increase in invasion was inhibited by re-expression of miR-200s while, in MDA-MB-231 cells, PGI/AMF-silencing-induced inhibition of invasion was eased by suppression of miR-200s.

Figure 5.

Figure 5

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miR-200s modulate PGI/AMF-mediated EMT and invasion of breast cells. (A) Western blot analysis for the expression of indicated EMT markers. β-actin protein was used as protein loading control. PM, specific pre-miRNAs (pre-miR200a + pre-miR200b + pre-miR200c); AM, specific anti-miRNAs (anti-miR200a + anti-miR200b + anti-miR200c); P/A, PGI/AMF-transfected cells; sP/A, PGI/AMF-silenced cells. (B) Invasion of breast cells MCF-10A (upper panel) and MDA-MB-231 (lower panel) was assayed by plating cells in matrigel-coated inserts. The cells that invaded through the matrigel were stained and photographed using fluorescence microscope. a, MCF-10A control cells; b, PGI/AMF- transfected MCF-10A cells; c, PGI/AMF- transfected MCF-10A cells + pre-miR200s (pre-miR200a + pre-miR200b + pre-miR200c); d, MDA-MB-231 control cells; e, PGI/AMF-silenced MDA-MB-231 cells and f, PGI/AMF-silenced MDA-MB-231 cells + anti-miR200s (anti-miR200a + anti-miR200b + anti-miR200c).

Role of miR-200s in experimental pulmonary metastases of breast cancer cells

Since our in vitro results suggested a mechanistic role of miR-200s in PGI/AMF-induced invasion, we carried out further investigations in vivo by injecting metastatic MDA-MB-231 cells via tail vein in SCID mice, which led to spontaneous lung metastases (Fig. 6) whereas either re-expression of miR-200 or silencing of PGI/AMF by siRNA led to significant decrease in pulmonary metastases (Fig. 6). The Wilcoxon tests comparing re-expression of miR-200 to control and silencing of PGI/AMF to control resulted p values of 0.0048 and 0.0043, respectively. Our in vitro experiments above suggested that loss of miR-200 is associated with increased tumor aggressiveness and that re-expression of miR-200 reduced tumor aggressiveness. Since PGI/AMF-silenced MDA-MB-231 cells showed reduced pulmonary metastases consistent with over-expression of miR-200, we hypothesized that anti-miR-200 could increase the pulmonary metastases of PGI/AMF-silenced MDA-MB-231 cells in vivo. We found that the treatment of animals, inoculated with PGI/AMF-silenced MDA-MB-231 cells, with anti-miR-200b [by intravenous injection with a special formulation that combines high affinity LNA-anti-miR with phosphorothioate modifications to achieve an efficient delivery and silencing of target miRNA (26)] led to increased pulmonary metastases (Fig. 6), suggesting a molecular link between PGI/AMF, miR-200 and metastases. The Wilcoxon test comparing non-specific anti-miRNA to specific anti-miRNA resulted p value of 0.0154.

Figure 6.

Figure 6

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Silencing of PGI/AMF suppresses lung metastasis and anti-miR-200 treatment abrogates this effect. (A) MDA-MB-231 cells (2 × 106 in a total volume of 0.1 ml) were injected via tail vein of ICR-SCID mice, and spontaneous pulmonary metastases were assessed after 5 weeks. The inserts on top of the box plot are representative lungs (left half piece) from each group with arrows indicating grossly visible tumors. (B) H&E staining revealed the extent of metastatic lesions in lungs. V, vector control; sP/A, PGI/AMF-silenced cells, NP, non-specific pre-miRNA; PM, specific pre-miRNAs (pre-miR200s); NA, non-specific anti-miRNA; AM, specific anti-miRNA (anti-miR200b). n=6 mice for each group.

Discussion

The major conclusions from our current study are: a) ectopic expression of PGI/AMF in non-tumorigenic MCF-10A breast epithelial cells results in the down-regulation of epithelial marker, E-cadherin, with concomitant up-regulation of mesenchymal markers vimentin, ZEB1, ZEB2, and NF-κB activity. Silencing of PGI/AMF in MDA-MB-231 cells, on the other hand, results in up-regulation of E-cadherin and down-regulation of vimentin, ZEB1, ZEB2, and NF-κB activity; b) expression of PGI/AMF correlates negatively with the expression of miRNAs 200a, 200b and 200c; c) re-expression of these miRNAs in MCF-10A cells abrogates the effects of PGI/AMF over-expression and restores epithelial phenotype while their silencing in MDA-MB-231 cells overcomes the changes brought about by PGI/AMF silencing; d) miRNAs play a key role in the PGI/AMF-mediated invasion of breast cells in vitro, pulmonary metastasis in vivo and our data suggests that the modulation of these miRNAs by PGI/AMF appears to be the mechanism by which PGI/AMF influences the aggressiveness of breast cancer cells.

AMF was originally isolated and purified from the conditioned medium of human melanoma cells A2058 (34). It was so named due to its ability to stimulate motility of the cells that produced/secreted it. AMF was also found to be produced by clones of ras-transfected metastatic NIH 3T3 cells (34). Characterization of AMF secreted by mouse fibrosarcoma cells Gc-4 PF revealed that it is phosphohexo isomerase/phosphogluco isomerase (EC 5.3.1.9) (2). We recently demonstrated the ability of PGI/AMF to induce EMT in MCF-10A cells (12). Also, silencing of PGI/AMF in MDA-MB-231 cells led to the reversal of EMT (12). In the present study, we report for the first time that PGI/AMF over-expression leads to increased expression of ZEB1 and ZEB2 in MCF-10A cells while its silencing results in their down-regulation in MDA-MB-231 cells. Since ZEB1 and ZEB2 are markers of mesenchymal phenotype, such results were expected; however, this observation, in combination with the knowledge that miR-200 family is known to regulate the processes of EMT by targeting ZEB1 and ZEB2, led us to hypothesize that miR-200 family plays a crucial role in PGI/AMF-induced EMT in breast cells.

To test our hypothesis, we assessed the expression of miR200a, miR200b and miR200c in paired cell lines – MCF-10A (vector vs. PGI/AMF-transfected) and MDA-MB-231 (vector vs. PGI/AMF-silencing). We found that PGI/AMF-transfection resulted in a significant down-regulation while silencing of PGI/AMF resulted in a significant induction of all these miRNAs. These results demonstrated, for the first time, the ability of PGI/AMF to modulate the expression of miRNAs. From the time of recognition of microRNAs as regulatory molecules less than a decade ago (35-37), there have been numerous reports on the involvement of miRNAs in the regulation of many disease conditions, including cancer. Independently, PGI/AMF (38) as well as various miRNAs (39) have been linked with the processes of invasion and metastasis of human breast cancer cells. The data presented here provides a molecular link between these two factors. Additionally, our in vivo studies clearly suggest a role of miR-200 family in PGI/AMF-induced pulmonary metastasis of breast cancer.

Transcription factor NF-κB is constitutively active in cancer cells and is therefore an attractive therapeutic target (40). We found an increased NF-κB activity in PGI/AMF over-expressing MCF-10A cells (Fig. 1), which also correlated with increased colonization (Fig. 4) and invasion (Fig. 5). Since activation of NF-κB has been linked to the induction of EMT (27-29, 41), increased activity of NF-κB could explain the induction of EMT in PGI/AMF over-expressing cells (12). It has previously been shown that NF-κB suppresses E-cadherin, and induces vimentin, leading to EMT in MCF-10A cells with constitutively active p65 subunit of NF-κB (27). Likewise, the levels of ZEB1 and ZEB2 were also found to be elevated in these cells, and the silencing of ZEB1 or ZEB2 inhibited the growth of these cells with no effect on parental cells, suggesting the importance of ZEB1/ZEB2 in cells with activated NF-κB. Our own experiments with MCF-10A cells are in full agreement with these reports because we found a positive correlation between the induction of EMT with increased DNA binding activity of NF-κB and increased expression of ZEB1 and ZEB2.

Regulation of oncogenes/tumor suppressor genes by miRNAs is increasingly being realized to be a key step in the progression of human malignancies (42). In the context of EMT induction, there are numerous reports regarding the involvement of miR-200 family in this process (22, 32, 33, 43). Earlier results from our laboratory have shown that the re-expression of miR-200 by pre-miR-200 transfection (23) or the treatment of cells by natural dietary agent (44) could inhibit aggressiveness and invasion characteristics of cancer cells. In this context, modulation of miR200s by PGI/AMF provides a mechanism through which PGI/AMF may influence the aggressiveness of breast cancer cells. Our in vitro assays such as wound healing and invasion assays together with in vivo experimental pulmonary metastases assay, provided direct evidence in support of the involvement of miR-200s in PGI/AMF-mediated effects on aggressive behavior of MDA-MB-231 cells. Using a reciprocal model in MCF-10A cells, we show that PGI/AMF-mediated induction of invasion can be effectively blocked by re-expression of miR-200s. Our results with the expression levels of EMT markers (Fig. 5) in the two cell lines confirm the key regulatory role of miR-200s in the modulation of EMT by PGI/AMF.

Based on existing literature together with our current findings, a model for the regulation of EMT by PGI/AMF involving miR-200s, NF-κB and mesenchymal markers ZEB1/ZEB2 is emerging as represented by a hypothetical diagram (Fig. 7). There seems to be a complex reciprocal relationship between miR-200s and ZEB1/ZEB2. It has been suggested that there are three putative binding sites for miR-200a in the 3′UTR region of ZEB1 as well as ZEB2 while miR-200b/c have five binding sites in the 3′UTR region of ZEB1 and six sites in ZEB2 (22), respectively. The miR-200s repress the expression of ZEB1 and ZEB2 by direct binding to these sites (22). Reciprocally, ZEB1 and ZEB2 negatively regulates miR-200a/b by binding to paired CACCTG (E-box) sites (45). Such repression has been demonstrated in several mesenchymal breast cancer cell lines, including those used here, MDA-MB-231 and BT-549 (45). Another report showed direct repression of miR200s by ZEB1, and a significant up-regulation of miR-200b/c following a stable knock-down of ZEB1 in MDA-MB-231 cells (46). Collectively, these reports indicate a reciprocal relationship between miR-200s and ZEB1/ZEB2 as summarized in Fig. 7. A direct relationship between miR-200s and NF-κB has not been clearly established; however, there is evidence to suggest the regulation of ZEB1/ZEB2 by NF-κB (27). In this report, ectopic expression of NF-κB (p65) in MCF-10A cells was shown to induce ZEB1/ZEB2 expression, and p65 was observed to regulate ZEB1 via direct binding to its promoter. Our results clearly show up-regulation of NF-κB (Fig 1C) and down-regulation of miR-200s (Fig 2 and 3) by PGI/AMF, and both these events could result in the up-regulation of ZEB1/ZEB2 leading to EMT and aggressiveness of cancer cells (Fig. 7), thus explaining the mechanism of PGI/AMF action.

Figure 7.

Figure 7

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Schematic representation of regulation of EMT and invasion of breast cancer cells by PGI/AMF. The miR200s seem to play a key role in the induction of EMT by PGI/AMF. The complex relationship between miR200s, NF-κB, ZEB1/ZEB2 and E-cadherin, and their regulation by PGI/AMF might be crucial to the acquisition of EMT and aggressive behavior of breast cancer cells.

Since metastasis of breast cancer is directly related to poor prognosis, thus molecular markers of invasion and metastasis, such as PGI/AMF, offer attractive targets for therapy. PGI/AMF-mediated induction of EMT provides a broad mechanism through which it influences the invasion of breast cells. Here, we provided further insight into the individual regulator, the miR-200 family, in mediating the effects of PGI/AMF signaling at the molecular level. These in vitro and in vivo results provides mechanistic evidence, for the first time, linking miR-200 with the biological activity of PGI/AMF, further suggesting that innovative approaches by which miR-200s could be up-regulated can potentially serve as a novel therapeutic strategy for the treatment of highly invasive breast cancer in the future.

Acknowledgments

This work was supported in part by NIH-NCI grant R01CA51714 (A Raz)

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